Abstract Allogeneic hematopoietic-cell transplantation (allo-HCT) is an important treatment with curative intent for hematological malignancies. Graft-vs-host disease (GVHD) is a major cause of morbidity and mortality after transplantation. In human analyses and animal experiments, we and others have shown that the intestinal microbiota contribute to the pathophysiology of GVHD, as well as post-transplant infections and relapse. Using 16S ribosomal RNA next-generation sequencing, we examined the intestinal microbiota of allo-HCT patients and found a post- transplant ?microbiota injury? with loss of diversity and a dramatic expansion of potentially pathogenic bacteria that precedes bacterial sepsis with the same organisms. This dysbiosis is likely due to the combined effects of (a) broad-spectrum antibiotics for the treatment of post- transplant febrile neutropenia and (b) the profound nutritional alterations experienced by these patients. We found an inverse relationship between a loss of the genus Blautia after allo-HCT and GVHD mortality. We observed that broad-spectrum antibiotics that target the anaerobic commensal flora are particularly associated with increases in GVHD-related mortality and in fact worsened intestinal GVHD in our animal model. We thus hypothesize that the intestinal microbiota has a significant role in the development of GVHD, is a potent modulator of GVHD severity, and can be targeted to prevent or treat GVHD. We propose studies that aim to elucidate the mechanisms by which the microbiota can regulate the pathophysiology of GVHD (and graft-vs- tumor (GVT) activity), as well as studies to translate these findings into clinical biomarkers and therapies for allo-HCT patients with GVHD. In Aim 1 we will assess the intestinal microbiota of allo-HCT patients for its relationship with acute and chronic GVHD. We will study the relationship of a) pre-HCT intestinal microbiota with and use as biomarkers for acute GVHD, and b) pre- and/or post-HCT microbiota with chronic GVHD. We will also study the recovery of the intestinal microbiota after allo-HCT and its relationship with immune reconstitution. In Aim 2 we will assess the mechanisms by which microbiota can influence alloreactivity and graft-versus-tumor activity in preclinical models and study the effects of individual microbes, defined bacterial ecologies and fecal microbiota transplantation on GVHD and GVT activity. These studies form the basis for ongoing and future trials targeting the intestinal microbiota to reduce GVHD and transplant-related mortality.